Research will continue on the long-term goal of elucidating the molecular architecture of alpha-crystallin, the major protein component of the mammalian lens, and defining the mechanism of its chaperone function. The specific goals for the next funding period are to undertake a detailed thermodynamic analysis of the interactions between alpha-crystallins and beta-crystallins, to investigate post-translational modifications that modulate these interactions, and to define the global structural and dynamic features of the native alpha-crystallin oligomer. The proposed research will definitively test the paradigm that alpha-crystallins maintain lens transparency through the recognition and binding of unfolding lens proteins. The results will bridge the knowledge gap between the rapidly developing understanding of the temporal trajectories of individual protein components and the consequences on their molecular interactions. The achievements of the previous funding period, including the determination of the folding pattern of the alpha-crystallin domain and the development of a mechanistic model of alphaA-crystallin chaperone function provide the bases for the proposed studies. Four specific aims are designed to address two fundamental questions: 1) What is the energy threshold required to trigger the binding of beta-crystallin to alpha-crystallin and is this threshold crossed in age-related modifications? 2) How is this interaction modulated by the co-oligomerization of alpha-crystallin subunits and by phosphorylation of alphaB-crystallin? For this purpose, site-directed mutants of selected beta-crystallins will be constructed and characterized with respect to their stability and binding affinity to alpha-crystallin. Structural analysis will be performed using newly developed Electron Paramagnetic Resonance distance measurement methods on the 50A scale. Phosphorylation-mimic substitutions will be introduced in alphabeta-crystallin and their effects on the structure, dynamics and binding to beta-crystallins investigated. A unique combination of molecular biology and biophysical methods will be used to address a problem of central importance to the understanding of the molecular basis of lens transparency. To the extent that modifications of beta-crystallins play a role in age-related opacity, the proposed studies are of fundamental biomedical importance. More generally, a growing number of pathologies have been traced to protein aggregation and failures of the chaperone machinery. The anticipated results will elucidate common molecular events associated with these diseases.